Composition for treating diabetes and dyslipidemia obtained from the extract of costus pictus d.don plant and a method of preparing the same

ABSTRACT

The disclosure provides a medicinal composition for treating diabetes and dyslipidemia obtained from the extract of  Costus pictus  D. Don. The extract of  Costus pictus  D. Don includes hydroxycinnamic acid derivatives and flavonol mono-, di- and triglycosides along with small amounts of free flavonols. The extract of  Costus pictus  D. Don has low levels of oxalic acid and/or oxalates. The hydroxycinnamic acid derivatives include caffeic acid, p-coumaric acid, ferulic acid and sinapic acid and flavonol mono-, di- and triglycosides which include quercetin, kaempferol and isorhamnetin with sugars glucose, galactose and rhamnose. Disclosure also provides a method of preparing the extract of  Costus pictus  D. Don of the same. The disclosure also provides a method of treating mammals including human beings for medical conditions such as diabetes; dyslipidemia and related conditions; for increasing anti oxidant potential; for the regeneration of pancreatic beta cells and increasing the insulin secretogogue effect.

This Application is a divisional of U.S. Appl. Ser. No. 14/577,794 filedDec. 19, 2014, which is a continuation of International ApplicationSerial No. PCT/IN2013/000382, filed Jun. 20, 2013, which claims priorityof Indian Provisional Application Serial No. 2470/CHE/2012, filed Jun.22, 2012, all of which applications are hereby incorporated byreference.

FIELD OF INVENTION

The disclosure relates to an extract of Costus pictus D. Don having lowoxalic acid and/or oxalates content and method of producing the saidextract and more specifically a composition for treating diabetes anddyslipidemia obtained from the extract of Costus pictus D. Don plant anda method of producing the same. The constituents of the said extract ofCostus pictus D. Don having low oxalic acid and/or oxalates contentcontaining hydroxycinnamic acid derivatives which include caffeic acid,p-coumaric acid, ferulic acid and sinapic acid and flavonol mono-, di-and triglycosides wherein said Flavonol mono-, di- and triglycosidesinclude quercetin, kaempferol and isorhamnetin with sugars glucose,galactose & rhamnose along with small amounts of free flavonols which isused for treating diabetes, dyslipidemia and related disorders. Theselipids include cholesterol, cholesterol esters, phospholipids, andtriglycerides. Lipids are transported in the blood as large‘lipoproteins’.

BACKGROUND

Diabetes mellitus was known to ancient Indians as early as sixth centuryB. C. Charaka in his “Charaka Samhita” has mentioned the sweetness ofurine in addition to polyuria. The Indian physician Susruta in 500 A. Ddescribed the disease as “Madhumeha” meaning rain of honey, (due to thephenomenon of attracting ants near the urine of a diabetic patient) withsymptoms of foul breath, voracious appetite and languor. Other earlyIndian writings like Astanga-hrayda, Bhava-prakasa, Madhav-nidana etc.have also described diabetes.

Globally, the number of people with diabetes is expected to rise fromthe current estimate of 150 millions to 220 millions in 2010 and 300millions in 2025. The prevalence is increasing in the developingcountries such as India, particularly in urban areas. The estimatednumber of diabetes patients in India was 19.4 million in 1995 and areexpected to be 57.2 million in 2025 (W.H.O). In the United States, it isestimated that as of 2011, 25.8 million people (8.3% of the totalpopulation) were diabetic. About 2.15 lakhs of people under 20 years ofage have diabetes (0.26% of all people in this age group). Approximatelyone in every 400-500 children and adolescents has Type-1 diabetes. Inthe age group of 20 years or older, 25.6 million (11.8% in men and 10.8%in women) have diabetes. In people above the age of 60 years, 10.9million (26.9% of all people in this age group) have diabetes.

There are mainly two types of diabetes. Type-1 diabetes which waspreviously called insulin dependent diabetes mellitus (IDDM) orjuvenile-onset diabetes. This develops when the body's immune systemdestroys pancreatic beta cells ((3-cells), which are the only cells inthe body that make the hormone insulin that regulates blood glucoselevel.

Type-II diabetes was previously called non-insulin dependent diabetesmellitus (NIDDM) or adult onset diabetes. It usually begins as insulinresistance, a disorder in which the cells of the body fails to respondto insulin properly.

Unlike what was happening a few decades ago, anti-diabetic herbalformulations are marketed now in plenty in the form of powders, tablets,capsule and liquid preparations. Most of these have not undergonecontrolled clinical trials. Usually herbs with reputed anti-diabeticactivity as documented in standard ayurvedic books or knowledge gainedfrom experienced Ayurvedic practitioners are taken up and mixtures areprepared with arbitrary dosage. It is doubtful how some of thesepreparations are effective and what are the side effects, on long termusage are.

The medicinal plant Costus pictus D. Don is a very popular andfast-spreading ginger belonging to the family of Zingiberaceae that hasbeen used as an ornamental climbing plant and is used as a munchingdietary supplement for the treatment of diabetes in southern India.Costus pictus D. Don, commonly called as Spiral ginger, Stepladder orInsulin plant, belongs to the family Costaceae. The plant is cultivatedas an ornamental plant in tropical gardens. It is a perennial herbgrowing up to 2-3 m and spreads 1.5-2 m. The glossy linear narrow leaveswith characteristic wavy edges are arranged spirally on red colouredstem. The leaves are less fleshy and have an acrid taste. Theinflorescences form both at the end of a leafy stem, and less oftenradically on a short nearly leafless stem. It can be recognized by itsyellow flowers with red spots and stripes and appear in terminal cone.Propagation is carried out through stem cuttings and rhizome.

Different extracts of Costus pictus D. Don obtained from various partsof Costus pictus D. Don is reported to have different properties.

Costus pictus D. Don extracts have high concentration of oxalic acid.Since oxalic acid is found in high concentration in Costus pictus D. Donand found to be toxic, we standardized a method of making Costus pictusD. Don extract with very low level of oxalic acid content.

Oxalic acid is an organic compound with the formula H₂C₂O₄. It is acolorless crystalline solid that dissolves in water to give colorlesssolutions. It is classified as a dicarboxylic acid.

Oxalic acid is reported to be toxic for oral administration (MerckIndex, thirteenth edition, page 1237,para 6980; The European Agency forthe evaluation of medicinal products veterinary medicines andinspections, Committee for veterinary medicinal products Dec.2003; Sideeffects of oxalic acid, Tracey Roizman D C,2011).

Costus pictus D. Don extract (alcoholic, aqueous and juice extracts ofleaves/whole plant) is reported to have anti diabetic property (U.S.Pat. No. 7,255,886, Merina Benny (2007); Nandhakumar Jothivel et al(2007); M A Jayasri et al (2008)).

Costus pictus D. Don is reported to contain oxalic acid (Camargo et al.2006; Moron et al. 2007). The leaves of Costus pictus D. Don are sourin taste due to the presence of high levels of oxalic acid in theleaves. (Rajendran Sathishraj et al, 2011).

Costus pictus D. Don extracts have high concentration of oxalic acid. Ifit is in small quantity it may not be that harmful and on the contrarymay be beneficial against diabetes (Oxalic acid—Induced Modification ofPostglycation Activity of Lysozyme and its Glycoforms (Hong Ying Gao etal, 2010). But Oxalic acid is known to produce Kidney stones.(Effect ofdietary oxalate and calcium on urinary oxalate and risk of formation ofcalcium oxalate kidney stones, Linda K Massey, 1993).

In view of the above, we have developed a method of preparing an extractof Costus pictus D. Don having low oxalic acid and/or oxalates contentand composition of Costus pictus D. Don extract having low oxalic acidand/or oxalates content. The disclosure not only provides a method ofpreparation of an extract of Costus pictus D. Don having low oxalic acidand/or oxalates content, but also a composition derived from therein.The constituents of the said extract of Costus pictus D. Don having lowoxalic acid and/or oxalates content contain hydroxycinnamic acidderivatives and flavonol mono-, di- and triglycosides along with smallamounts of free flavonols which is used for treating diabetes,dyslipidemia and related disorders.

SUMMARY

Disclosure provides an extract of Costus pictus D. Don. Constituents ofthe extract include hydroxycinnamic acid derivatives and flavonols. Insome embodiments of the extracts, the total content of the oxalic acidand oxalates is less than about 15%. The disclosure provides an extractof Costus pictus D. Don having low oxalic acid/oxalate content as wellas a medical composition useful for treatment of diabetes; treatment ofdyslipidemia and related conditions; for increasing anti oxidantpotential; for the regeneration of pancreatic beta cells and increasingthe insulin secretogogue effect for mammals including human beings.

Further the disclosure also provides a method of preparation of anextract of Costus pictus D. Don having low oxalic acid/oxalates contenthaving the said medicinal composition.

Disclosure also discloses a method of treating mammals including humanbeings for medical conditions selected from diabetes; dyslipidemia andrelated conditions; for increasing antioxidant potential; for theregeneration of pancreatic beta cells and increasing the insulinsecretogogue effect.

In some embodiments, the constituents of hydroxycinnamic acidderivatives include caffeic acid at a concentration of about 0.1% andabove, p-coumaric acid at a concentration of about 0.1% and above,ferulic acid at a concentration of about 0.1% and above and sinapic acidat a concentration of about 0.1% and above. Some embodiments, of theextract also contain components as acid derivatives, glucosides or infree form. Some embodiments include flavonol mono-, di- andtriglycosides which include quercetin at a concentration of about 0.1%and above, kaempferol at a concentration of about 0.1% and above andisorhamnetin at a concentration of about 0.1% and above with sugarsglucose, galactose and rhamnose. In addition it also contains smallamounts of free flavonols comprises quercetin at a concentration ofabout 0.1% and above, kaempferol at a concentration of about 0.1% andabove and isorhamnetin at a concentration of about 0.1% and above.

The extract of Costus pictus D. Don can be derived from fresh wholeplant, fresh leaves, dried leaves or dried leaf powder, juice of freshleaves, fresh or dried aerial parts of Costus pictus D. Don orcombinations thereof.

The method of producing the said composition includes the steps of a)selecting the raw material and cleaning to make it free of any foreignmatter like dirt b) cutting the cleaned material into small pieces c)extraction with solvents selected from water, hexane, methanol, ethanol,isopropanol, n-butanol, methyl acetate, ethyl acetate, propyl acetate,n-butyl acetate and combinations thereof to obtain an extract d) removalof oxalic acid from the extract by heating the extract with water at 80°C. e) purifying and concentrating the resultant extract and dry toobtain in dry powder form.

Another embodiment provides extract of Costus pictus D. Don preparedfrom fresh leaves of Costus pictus D. Don by extraction with 90%methanol and oxalic acid content in the extract is reduced by heatingthe extract with water at 80° C.

Yet another embodiment provides a methanol extract of Costus pictus D.Don having low oxalic acid and/or oxalates content is purified bypassing through a silica column.

In another embodiment methanol extract of Costus pictus D. Don havinglow oxalic acid and/or oxalates content is again extracted with ethylacetate.

In general said composition, the extract of Costus pictus D. Don will behaving an oxalic acid and/or oxalates content as follows:

-   -   a) about 10% and below, if it is a methanol extract;    -   b) about 5% and below, if said methanol extract is further        purified;    -   c) about 2.5% and below, if it is an ethyl acetate extract;    -   d) about 1% and below, if it is a purified ethyl acetate        extract.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the disclosure should become apparentfrom the following description and read in conjunction with the figuresof the accompanying drawings, which demonstrate the findings of variousprocess trials carried under certain embodiments under the disclosure aswell as the findings.

FIG. 1 shows chromatogram peaks corresponding to hydroxycinnamic acidderivatives at 254 nm on High performance liquid chromatography (HPLC)of a 90% methanol extract of fresh leaves of Costus pictus D. Don, andwherein the extract had low oxalic acid and/or oxalates content (sample2 prepared as per example 1).

FIG. 2 shows peaks corresponding to flavonoids on High performanceliquid chromatography (HPLC) chromatogram at 324 nm of a 90% methanolextract of fresh leaves of Costus pictus D. Don, wherein the extract hadlow oxalic acid and/or oxalates content (sample 2 prepared as perexample 1).

FIG. 3. Provides High performance liquid chromatography (HPLC) data ofalkali hydrolysis product of 90% methanol extract (having low oxalicacid and/or oxalates content) of fresh leaves of Costus pictus D. Don(sample 2 prepared as per example 1) in which hydroxycinnamic acids(p-coumaric acid, caffeic acid, ferulic acid and sinapic acid) weredetected at 324 nm and identified by co-injection with referencestandards.

FIG. 1 a. provides HPTLC finger print in visible light of 90% methanolextract (having low oxalic acid and/or oxalates content) of fresh leavesof Costus pictus D. Don (sample 2 prepared as per example 1) afteralkali hydrolysis shows the presence of p-coumaric acid, caffeic acid,ferulic acid and sinapic acid.

FIG. 4b -provides fingerprinting shows the presence of p-coumaric acid,caffeic acid, ferulic acid and sinapic acid at in visible UV light onHPTLC of alkali hydrolysed 90% methanol extract of fresh leaves ofCostus pictus D. Don (sample 2 prepared as per example 1), the extracthad low oxalic acid and/or oxalates content.

FIG. 5. shows flavonols (quercetin, kaempferol and isorhamnetin) peaksdetected at 368 nm on High performance liquid chromatography (HPLC)analysis of acid hydrolysed 90% methanol extract of fresh leaves ofCostus pictus D. Don (sample 2 prepared as per example 1), the extracthad low oxalic acid and/or oxalates content.

FIG. 6 a. shows the presence of sugars (glucose, galactose and rhamnose)by High performance thin layer chromatography (HPTLC) at visible lightof acid hydrolysed 90% methanol extract of fresh leaves of Costus pictusD. Don, the extract had low oxalic acid and/or oxalates content (sample2 prepared as per example 1).

FIG. 6b shows the presence of sugars (glucose, galactose and rhamnose)on High performance thin layer chromatography (HPTLC) at UV light ofacid hydrolysed 90% methanol extract of fresh leaves of Costus pictus D.Don, the extract had low oxalic acid and/or oxalates content (sample 2prepared as per example 1).

FIG. 7 provides Photodiode array detector (PDA) plots at 324 nm from theLCMS (Liquid chromatography-mass spectrometry) run of 90% methanolextract of fresh leaves of Costus pictus D. Don, the extract had lowoxalic acid and/or oxalates content (sample 2 prepared as per example1).

FIG. 8 provides Photodiode array detector (PDA) plots at 370 nm from theLCMS (Liquid chromatography-mass spectrometry) run of 90% methanolextract of fresh leaves of Costus pictus D. Don, the extract had lowoxalic acid and/or oxalates content (sample 2 prepared as per example1).

FIG. 9. shows primarily hydroxycinnamic acid derivatives on a photodiodearray detector (PDA) plot at 324 nm from LCMS (Liquidchromatography-mass spectrometry) run of 90% methanol extract of freshleaves of Costus pictus D. Don, and the extract had low oxalic acidand/or oxalates content (sample 2 prepared as per example 1).

FIG. 10. shows presence of flavonoid derivatives on Photodiode arraydetector (PDA) plot at 370 nm from LCMS (Liquid chromatography-massspectrometry) run of 90% methanol extract of fresh leaves of Costuspictus D. Don, the extract had low oxalic acid and/or oxalates content(sample 2 prepared as per example 1).

FIG. 11. Histopathological examination of the pancreas of normal group.The marked area depicts an islet of Langerhans with the beta cellsstained brown.

FIG. 12. Histopathological examination of the pancreas of Diabeticcontrol group. The marked area depicts an islet of Langerhans with veryfew beta cells.

FIG. 13. Histopathological examination of the pancreas of Glibenclamidetreated group. The marked area depicts an islet of Langerhans withmoderately stained beta cells.

FIG. 14. Histopathological examination of the pancreas of group treatedwith methanolic extract of fresh leaves of Costus pictus D. Don, theextract had low oxalic acid and/or oxalates content (Sample 2 preparedas per Example 1). The marked area depicts an islet of Langerhans withsparsely stained beta cells.

FIG. 15. Histopathological examination of the pancreas of group treatedwith silica purified ethyl acetate extract of 90% methanolic extract offresh leaves of Costus pictus D. Don, and the extract had low oxalicacid and/or oxalates content (Sample 5 prepared as per Example 4). Themarked area depicts an islet of Langerhans with intensely stained andincreased number of beta cells.

FIG. 16. Histopathological examination of the pancreas of group treatedwith Ethyl acetate extract of 90% methanol extract of fresh leaves ofCostus pictus D. Don, the extract had low oxalic acid and/or oxalatescontent (Sample 4 prepared as per Example 3). The marked area depicts anislet of Langerhans with moderately stained beta cells.

FIG. 17. Histopathological examination of the pancreas of group treatedwith methanolic extract of fresh leaves of Costus pictus D. Don (Sample1 prepared as per Example 1). The marked area depicts an islet ofLangerhans with moderately stained and increased number of beta cells.

DETAILED DESCRIPTION

The disclosure provides an extract of Costus pictus D. Don having lowoxalic acid and/or oxalates content useful for the treatment ofdiabetes, treatment of dyslipidemia and related conditions. Thedisclosure also provides composition of Costus pictus D. Don extracthaving low oxalic acid and/or oxalates content and a method ofpreparation of an extract of Costus pictus D. Don having low oxalic acidand/or oxalates content.

The present disclosure provides an extract of Costus pictus D. Donhaving low oxalic acid and/or oxalates content. The extract containshydroxycinnamic acid derivatives and flavonol mono-, di- andtriglycosides along with small amounts of free flavonols.Hydroxycinnamic acid derivatives include caffeic acid, p-coumaric acid,ferulic acid and sinapic acid. The flavonols include free flavonols suchas quercetin, kaempferol, isorhamnetin. Flavonols also include glucosederivatives of quercetin, glucose derivatives of kaempferol, glucosederivatives of isorhamnetin, galactose derivatives of quercetin,galactose derivatives of kaempferol, galactose derivatives ofisorhamnetin, rhamnose derivatives of quercetin, rhamnose derivatives ofkaempferol, and rhamnose derivatives of isorhamnetin. Flavonols includeflavonol mono-, di- and triglycosides with sugars such as glucose,galactose and rhamnose. In some embodiments, the constituents offlavonols include small amounts of free flavonols such as quercetin,kaempferol and isorhamnetin.

We found that 90% methanolic extract of Costus pictus D. Don extract hasabout 56 to 60% oxalic acid and or oxalates.

Costus pictus fresh leaves contain oxalic acid and oxalates. (Oxalicacid and oxalate oxidase enzyme in Costus pictus D. Don, RajendranSathishraj, Antoney Augustin, Acta Physiol plant, DOI 10.1007/s11738-011-0866-x.) Fresh leaves contain 5% w/w oxalic acid and driedleaves contain 1% w/w oxalic acid. The oxalates found in leaves aresoluble oxalates (sodium oxalate, potassium oxalate) and insolubleoxalate (calcium oxalate). Soluble oxalates are soluble in methanol andwater, but calcium oxalate is not soluble water. (The probable functionof calcium oxalate crystals in plants, Alberts Schneider, Chicagojournals, Botanical Gazette, Vol.32, No.2 (Aug.1901), pp. 142-144.)Oxalic acid is freely soluble in water. In the disclosed embodiments,when the Costus pictus leaves are extracted with methanol, the methanolextract contains methanol soluble and water soluble oxalates such assodium oxalate, potassium oxalate, and oxalic acid. The methanol extractis then treated with water and water part (supernatant) is removed toobtain water insoluble part of methanol extract of fresh leaves ofCostus pictus. Treatment with water removes sodium oxalate, potassiumoxalate, oxalic acid in to the water part (supernatant). The oxalic acidcontent as analysed by HPLC in methanol extract is 56%, in waterinsoluble part it is 8% and water part it is 75%. The HPLC analysis foroxalic acid comprises treating the extract with H2504, which againhydrolyses and oxalates present in the extract to liberate free oxalicacid.

Since oxalic acid is found in high concentration in Costus pictus D. Donand it is found to be toxic, we standardized a method of making Costuspictus D. Don extract with very low level of oxalic acid and oroxalates. The new extract was tested for its anti diabetic action and wefound that anti diabetic activity of Costus pictus extract did notdecrease after reducing the oxalic acid or oxalates content in theextract.

The disclosure provides a dosage form of composition of an extract ofCostus pictus D. Don having low oxalic acid and/or oxalates content. Thedisclosure provides a dosage form of composition of an extract of Costuspictus D. Don having low oxalic acid and/or oxalates content for oraladministration. Dosage forms of the extract are selected from the groupconsisting of a capsule, tablet, granule, sachet, powder, paste,ointment, infusion, injection, ampoule, solution, suspension, emulsion,pill, sustained release formulation and combinations thereof. In someembodiments, the dosage forms include fillers. Fillers can be lactose,spray dried lactose, starch, dibasic calcium phosphate, tribasic calciumphosphate, microcrystalline cellulose, hydroxy propyl methyl cellulose,calcium carbonate or combinations thereof.

The disclosed composition of extract Costus pictus D. Don is useful forthe treatment of diabetes, dyslipidemia and is also useful forincreasing antioxidant potential in mammal. Mammals includestreptozotocin induced diabetic rats and humans. The method of treatmentof diabetes mellitus and dyslipidemia includes administering a dose ofabout 50 mg/kg/day to about 200 mg/kg/day of composition of an extractof Costus pictus D. Don having low oxalic acid and/or oxalates contentto the streptozotocin induced diabetic rat. The method of treatment ofdiabetes mellitus and dyslipidemia includes administering a dose ofabout 500 mg/day to about 2000 mg/day of composition of an extract ofCostus pictus D. Don having low oxalic acid and/or oxalates content to ahuman.

In one embodiment the disclosure provides an extract of Costus pictus D.Don having low oxalic acid and/or oxalates content wherein thecomponents can be derived from fresh whole plant, fresh leaves, driedleaves or dried leaf powder, juice of fresh leaves, fresh or driedaerial parts of Costus pictus D. Don or combinations thereof.

The disclosure provides constituents of an extract of Costus pictus D.Don having low oxalic acid and/or oxalates content wherein the extractis obtained by treatment with low molecular weight alcohols, as such ormixed with water, halogenated hydrocarbons, organic ethers, lowmolecular weight esters, other organic solvents and low molecular weightketones.

Low molecular weight alcohols that can be used in preparation of theextract include methanol, ethanol, isopropanol, n-butanol andcombinations thereof. Halogenated hydrocarbons that can be used forextract preparation include methylene chloride, ethylene dichloride,chloroform, and combinations thereof. Esters that can be used forextract preparation include methyl acetate, ethyl acetate, propylacetate, n-butyl acetate and combinations thereof. Ketones that can beused for extract preparation include acetone, methyl ethyl ketone, andcombinations thereof. Alkanes that can be used for preparation of theextract include pentane, hexane, heptane, isooctane, and combinationsthereof.

Various methods for the preparation of extract of Costus pictus D. Donprepared by the extraction of fresh leaves of Costus pictus D. Don areprovided.

One of the embodiments under the disclosure is the extraction of freshleaves of Costus pictus D. Don with 90% methanol where oxalic acid isremoved from the extract by heating the extract with water at 80° C. Theresultant dried powder of 90% methanol extract of Costus pictus D. Donhas low oxalic acid and/or oxalates content. The process steps involvedare as under.

Fresh leaves of Costus pictus D. Don are extracted with 90% methanol.The mixture is refluxed for one hour to obtain a first residue andsupernatant. After first extraction, the first residue is furtherextracted two more times with four times the quantity of methanol ateach time. The residue and supernatants are separated. All thesupernatants are pooled and concentrated to form concentrated methanolextract. Concentrated methanol extract is dried to get powder of 90%methanolic extract of Costus pictus D. Don. Powder of 90% methanolicextract of Costus pictus D. Don is heated with water at 80° C. Firstresidue and water part is obtained and the water part is discarded.First residue is again heated with water at 80° C. Second residue andwater part is obtained. While the water part is discarded, secondresidue is dried to get powder of 90% methanol extract of fresh leavesof Costus pictus D. Don. The extract has low oxalic acid and/or oxalatescontent (8%).

Another embodiment provides method of preparation of purified 90%methanol extract of fresh leaves of Costus pictus D. Don. The resultingextract has low oxalic acid and/or oxalates content. Powder of 90%methanol extract of fresh leaves of Costus pictus D. Don is dissolved inminimum amount of solvent and silica gel is added in a vessel. Swirl themixture until the solvent evaporates and passed through a column, whichis already wet packed with silica gel and hexane. Then it is eluted withhexane followed by methanol. Methanol fractions are collected and hexanefraction is discarded. Methanol fraction is concentrated and dried toget purified 90% methanol extract of fresh leaves of Costus pictus D.Don, and the extract has low oxalic acid and/or oxalates content (3%).

One embodiment provides a method of preparing ethyl acetate extract of90% methanol extract of fresh leaves of Costus pictus D. Don, and theextract has low oxalic acid and/or oxalates content. Fresh leaves ofCostus pictus D. Don are extracted with 90% methanol to get powder of90% methanolic extract of Costus pictus D. Don. Powder is treated withwater and heated at 80° C. to remove the oxalic acid content to getpowder of 90% methanol extract of fresh leaves of Costus pictus D. Don,and the extract has low oxalic acid and/or oxalates content. Powder of90% methanol extract of fresh leaves of Costus pictus D. Don, and wherethe extract has low oxalic acid and/or oxalates content, is dissolved inwater and is extracted with ethyl acetate and subsequent separation oftwo phases-aqueous and ethyl acetate. Ethyl acetate part is collected,concentrated and dried to form powder of ethyl acetate extract of 90%methanol extract of Costus pictus D. Don. The extract has low oxalicacid and/or oxalates content (1.5%). The water phase is discarded.

Another embodiment provides a method of preparing silica purified ethylacetate extract of 90% methanol extract of fresh leaves of Costus pictusD. Don, wherein the extract has low oxalic acid and/or oxalates content.Ethyl acetate extract of 90% methanol extract of fresh leaves of Costuspictus D. Don wherein the extract has low oxalic acid and/or oxalatescontent is dissolved in minimum amount of hexane and silica gel is addedin a vessel. Swirl the mixture until the solvent evaporates and passedthrough the column, which is already wet packed with silica gel andhexane. Then it is eluted with hexane followed by methanol. Methanolfractions are collected and hexane fraction is discarded. Methanolfractions are concentrated and dried to get purified ethyl acetateextract of 90% methanol extract of fresh leaves of Costus pictus D. Donand the extract has low oxalic acid and/or oxalates content (0.2%).

Some embodiments provide a method of preparing the extract of Costuspictus D. Don. The method includes cleaning a starting material ofCostus pictus D. Don to obtain a cleaned material. The starting materialof Costus pictus D. Don can be fresh whole plant, fresh leaves, driedleaves or dried leaf powder, juice of fresh leaves, fresh or driedaerial parts of Costus pictus D. Don or combinations thereof. Next thecleaned material is cut. Next, the cut material is extracted with asolvent to obtain a residue and a supernatant. The solvent can be water,hexane, methanol, ethanol, isopropanol, n-butanol, methyl acetate, ethylacetate, propyl acetate, n-butyl acetate or combinations thereof.Supernatant is concentrated and dried to form a dried powder. Driedpowder is heated with water at 80° C. to form second residue and secondsupernatant (water part). Water part contains oxalic acid and oxalates.The heating increases solubility of oxalic acid and oxalates in water.Therefore, decreases oxalic acid and oxalates content in the secondresidue. Next, the second residue is dried to obtain a dry powder. Thedry powder is dissolved in water and extracted with a solvent such aswater, hexane, methanol, ethanol, isopropanol, n-butanol, methylacetate, ethyl acetate, propyl acetate, n-butyl acetate or combinationsthereof to obtain aqueous phase and solvent phase. Solvent phase isconcentrated and dried to form powder. Powder is mixed with anothersolvent to form a mixture. Solvent can be water, hexane, methanol,ethanol, isopropanol, n-butanol, methyl acetate, ethyl acetate, propylacetate, n-butyl acetate or combinations thereof. Silica gel is added tothe mixture and resulting product is dried to form dried powder. Thedried powder is loaded onto a silica gel column. The silica gel columnis eluted with a solvent to obtain an eluate. Solvent can be water,hexane, methanol, ethanol, isopropanol, n-butanol, methyl acetate, ethylacetate, propyl acetate, n-butyl acetate or combinations thereof. Theeluate is concentrated and dried to obtain a dried powder extract ofCostus pictus D. Don.

Some embodiments provide a method of preparing an extract of Costuspictus D. Don. The method includes cleaning a starting material ofCostus pictus D. Don to obtain a cleaned material. The cleaned materialis cut. The cut product is extracted with 90% methanol to form amixture. The mixture is refluxed at the boiling temperature (60-70° C.)of methanol to obtain a residue and a supernatant. The residue isseparated from the supernatant by filtration. The supernatant isconcentrated to obtain a concentrated methanol extract. The concentratedmethanol extract is dried to obtain a powder of the methanol extract.The dried powder is mixed with water to obtain a mixture. The mixture isheated to obtain a residue and a supernatant. The residue andsupernatant are separated. The residue is dried to obtain a dry powderhaving about 10% of oxalic acid. Some embodiments of the dry powder haveless than 10% oxalic acid. Some embodiments of the dry powder have about8% oxalic acid. Some embodiments of the method further include mixingthe dry powder with methanol and silica gel to obtain a mixture. Thenevaporating methanol from the mixture to obtain a methanol treated drypowder which includes the extract and silica gel components. The drypowder is loaded onto a wet packed silica gel column to obtain anextract loaded silica gel column. The wet packed silica gel has silicagel and hexane. The extract loaded silica gel column is eluted withhexane to obtain a silica gel column after hexane elution. The silicagel column eluted with hexane is further eluted with methanol and amethanol eluate fraction is obtained. The methanol eluate fraction isconcentrated to form a concentrated extract. The concentrated extract isdried to obtain a dry powder having about 5% oxalic acid. Someembodiments of the dry powder have less than 5% oxalic acid. Someembodiments of the dry powder have about 3% oxalic acid.

Some embodiments provide a method of preparing an extract of Costuspictus D. Don. The method includes cleaning a starting material ofCostus pictus D. Don to obtain a cleaned material. The cleaned materialis cut. The cut product is extracted with 90% methanol to form amixture. The mixture is refluxed at the boiling temperature (60-70° C.)of methanol to obtain a residue and a supernatant. The residue isseparated from the supernatant by filtration. The supernatant isconcentrated to obtain a concentrated methanol extract. The concentratedmethanol extract is dried to obtain a powder of the methanol extract.The dried powder is mixed with water to obtain a mixture. The mixture isheated to obtain a residue and a supernatant. The residue andsupernatant are separated. The residue is dried to obtain a dry powderhaving about 10% of oxalic acid. Some embodiments of the dry powder haveless than 10% oxalic acid. Some embodiments further include dissolvingthe dry powder in water to obtain a mixture. Extracting the mixture withethyl acetate to obtain an aqueous phase and an ethyl acetate phase.Next concentrating the ethyl acetate phase to obtain a concentratedethyl acetate extract. Next, drying the ethyl acetate extract undervacuum at above 500 mm of mercury to form a dry powder of the ethylacetate extract. The dry powder has about 2.5% of oxalic acid. Someembodiments of the dry powder have less than 2.5% oxalic acid. Someembodiments of the dry powder have about 1.5% oxalic acid. Someembodiments of the method further include mixing the dry powder havingabout 2.5% of oxalic acid with methanol and silica gel to obtain amixture. Then drying the mixture to obtain a dry powder. Next the drypowder is loaded onto a wet packed silica gel column to obtain anextract loaded silica gel column. The wet packed silica gel column hassilica gel column in hexane solvent. The extract loaded silica gelcolumn is eluted with hexane to obtain a silica gel column after hexaneelution. The silica gel column eluted with hexane is eluted withmethanol and a methanol eluate fraction is obtained. The methanol eluatefraction is concentrated form a concentrated extract. The concentratedextract is dried to obtain a dry powder. Some embodiments of the drypowder have about 1% oxalic acid. Some embodiments of the dry powderhave less than 1% oxalic acid. Some embodiments of the dry powder haveabout 0.2% oxalic acid.

Some embodiments provide a composition having an extract of Costuspictus D. Don such as a methanol extract of Costus pictus D. Don havingabout 10% of oxalic acid. Some embodiments provide a composition havingan extract of Costus pictus D. Don such as a methanol extract of Costuspictus D. Don having about 5% of oxalic acid. Some embodiments provide acomposition having an extract of Costus pictus D. Don such as an ethylacetate extract of Costus pictus D. Don having about 2.5% of oxalicacid. Some embodiments provide a composition having an extract of Costuspictus D. Don such as an ethyl acetate extract of Costus pictus D. Donhaving about 1% of oxalic acid. Some embodiments provide an extract ofCostus pictus D. Don having a total of oxalic acid and oxalates contentranging from about 0.2% to about 10%.

Some embodiments are useful for treatment of diabetes, treatment ofdyslipidemia and related conditions by administering extract to amammal. Mammals include animals and human beings. Some embodimentsprovide a method of decreasing blood glucose level by administering theextract of Costus pictus D. Don. Some embodiments provide a method ofdecreasing HbAlc (glycosylated hemoglobin) by administering the extractof Costus pictus D. Don. Some embodiments provide a method of increasingserum insulin level by administering the extract of Costus pictus D.Don. Some embodiments provide a method of increasing liver and muscleglycogen. Some embodiments provide a method of increasing insulinsecretaegogue effect. Some embodiments provide a method of treatingdyslipidemia. Some embodiments provide a method of decreasingtriglycerides level. Some embodiments provide a method of decreasing LDLcholesterol levels. Some embodiments provide a method of decreasing VLDLcholesterol level. Some embodiments provide a method of increasing HDLcholesterol level. Some embodiments provide a method for increasing antioxidant potential. Some embodiments provide method of decreasing TBARSlevel (Thiobarbituric acid reactive substances). Some embodimentsprovide a method of increasing SOD level (Superoxide dismutase). Someembodiments provide a method of increasing catalase level. Someembodiments provide a method of increasing GSH level (Glutathione). Someembodiments provide a method of increasing GPx (glutathione peroxidase)level. Some embodiments provide a method for regeneration of pancreaticbeta cells.

Extract of Costus pictus D. Don having low oxalic acid contentcontaining hydroxycinnamic acid derivatives and flavonol mono-, di- andtriglycosides along with small amounts of free flavonols are identifiedby HPLC and LCMS analysis.

The extract of Costus pictus D. Don is analyzed by HPLC for the presenceof hydroxy cinnamic acid and flavonoids.

The UV data at 254 nm (FIG. 1) shows several peaks corresponding tohydroxycinnamic acid. UV data at 324 nm (FIG. 2) shows several peakswhich corresponding to flavonoids.

Alkali hydrolysis and acid hydrolysis of the extract of Costus pictus D.Don confirm the presence of hydroxycinnamic acids and flavonoidsrespectively. The HPLC analysis of alkali hydrolysed extract of Costuspictus D. Don detects hydroxycinnamic acid at 324 nm. Accordingly,p-coumaric acid, caffeic acid, ferulic acid and sinapic acid areidentified after alkali hydrolysis of the extract (FIG. 3) byco-injection with reference standards.

HPTLC finger print (FIG. 4a and FIG. 4b ) of Costus pictus D. Donextract after hydrolysis shows the presence of p-coumaric acid, caffeicacid, ferulic acid and sinapic acid (Track 5) against referencestandards ferulic acid (Track 1), Sinapic acid (Track 2), p-coumaricacid(Track 4) and caffeic acid (Track 5).

Similarly HPLC analysis of acid hydrolysed extract of Costus pictusdetects flavonoids and the three peaks detected at 368 nm (FIG. 5) andthese corresponds to quercetin, kaempferol and isorhamnetin as confirmedby co-injection with reference standards.

HPTLC finger print (FIG. 6a and FIG. 6b ) of aqueous layer of Costuspictus D. Don extract after acid hydrolysis shows the presence of sugarslike glucose, galactose and rhamnose (Track 4) against referencestandards Glucose (Track 1), Ribose (Track 2), D-Galactose(Track 3),D-fructose (Track 4) and D-Rhamnose (Track 7).

The extract of Costus pictus D. Don is subjected to liquidchromatography-mass spectrometry (LCMS) analysis. The chromatograms at324 nm and 370 nm correspond to hydroxycinnamic acid derivatives andflavonoid glycosides respectively.

The LCMS chromatogram at 324 nm (FIG. 9) clearly shows a number ofcomponents, mainly hydroxycinnamic acid derivatives and these could beidentified from the MS data (Table 4).

In the same way flavonoids are detected at 370 nm by LCMS (FIG. 10). Thedata shows the presence of several flavonoid derivatives and these couldbe detected and identified. (Table 5)

It will be readily understood by the skilled artisan that numerousalterations may be made to the examples and instructions given hereinincluding alternate compositions without departing from either thespirit or scope of the present invention. These and other objects andfeatures of present invention will be made apparent from the followingexamples. The following examples as described are not intended to beconstrued as limiting the scope of the present invention.

EXAMPLE 1

Fresh leaves of Costus pictus D. Don were collected (5000 Kg). Freshleaves were cleaned and cut into small pieces. 90% methanol in an amountfour times the quantity of fresh leaves of Costus pictus D. Don wasadded to fresh leaves for methanol extraction. The extraction wasperformed using an extractor with reflux condenser. The bottom of theextractor was fitted with a polypropylene (100 microns) filter cloth.The mixture was refluxed at the boiling temperature (60-70° C.) ofmethanol for one hour to obtain a first residue and supernatant. Thefirst residue was then further extracted two more times with four timesthe quantity of methanol at each time. The residue and supernatants wereseparated by draining out the supernatant from the extractor bottomthrough the polypropylene filter cloth using a centrifugal pump. All thesupernatants were pooled and concentrated in an Agitated thin filmevaporator (ATFE) to form a concentrated methanol extract. Theconcentrated methanol extract was dried under vacuum at above 500 mm ofmercury to get dry powder of 90% methanol extract of fresh leaves ofCostus pictus D. Don (Yield 125 Kg).(sample 1). Now the oxalic acidcontent in 90% methanolic extract of Costus pictus D. Don is 56%.

Powder of 90% methanolic extract of Costus pictus (75 Kg of sample 1)was heated with water to form a residue and a supernatant (water part).Discard the water part. Residue was again treated with water and heat toform another residue and supernatant. Residue was dried under vacuum atabove 500 mm of mercury to obtain a dry powder. The yield of the powderwas 34 kg from a starting material of 75 kg. This powder was referred toas powder of 90% methanol extract of Costus pictus D. Don. This extracthas low oxalic acid and/or oxalates content. (Sample 2). The oxalic acidcontent in 90% methanolic extract of Costus pictus D. Don is only 8%.

Analysis of Oxalic acid by HPLC

Oxalic acid was estimated by high performance liquid chromatography(HPLC-DAD) on a C18 column (250×4.6 mm, Gemini 5 μm, USA.). The mobilephase was 5 mM H₂SO₄ and used under isocratic condition with an eluentflow rate of 1 ml/min. Oxalic acid was detected at 210 nm.

Standard was prepared by weighing 5 mg of standard oxalic acid (95%purity) and was made up to 50 ml with 6 mM H₂SO₄. Sample was prepared byweighing 50 mg of the dry extract of Costus pictus and was made up to 50ml with 6 mM H₂SO₄. Both the sample and standard were filteredseparately through a 0.2 μm membrane filter before injection into theHPLC column. The injection volume was 20 μl. Oxalic acid was detected at210 nm. By comparing the area of standard and sample, the percentage ofoxalic acid present in the sample was quantified.

${{Oxalic}\mspace{14mu} {acid}\mspace{14mu} \%} = {\frac{{Area}\mspace{14mu} {of}\mspace{14mu} {sample} \times {amount}\mspace{14mu} {of}\mspace{14mu} {standard}}{{Amount}\mspace{14mu} {of}\mspace{14mu} {sample} \times {Area}\mspace{14mu} {of}\mspace{14mu} {standard}} \times {purity}\mspace{14mu} {of}\mspace{14mu} {standard}}$

HPLC Analysis

50 mg of the Sample 2 was weighed and transferred into 50 ml standardflask and dissolved in methanol and made up to the mark using methanol.

The Sample 2 dissolved in methanol was analyzed on a Shimadzu ProminenceHPLC using a Phenomenex C-18 column (250×4.6 mm), 5 μ, RT (25° C.).

A gradient elution using solvents 5% ACN(acetonitrile) in water (solventA) and 95% ACN (acetonitrile) in water (solvent B) having 5 mM ammoniumacetate (pH 4) was used. The composition of solvent B was changed from5% to 50 in 35 min, then to 80% in 45 min and maintained at 80% till 55min and then maintained at 5% from 56 to 60 min. A UV detector at 254 nmand 324 nm was used to collect the data.

The UV data at 254/324 nm shows several peaks which correspond tohydroxycinnamic acid derivatives and flavonoids. Alkali hydrolysis andacid hydrolysis confirmed the presence of hydroxycinnamic acids andflavonoids respectively.

Acid Hydrolysis

150 g of Sample 2 was weighed and transferred into a glass lined vessel.Then Sample 2 was dissolved in 3.75 L of water and 562.5 ml concentratedhydrochloric acid was added to the vessel and was refluxed for 3 hours.The contents of the vessel were cooled and transferred into aliquid-liquid extractor and extracted with ethyl acetate. Ethyl acetatephase and aqueous phase were separated and ethyl acetate phase wascollected. Ethyl acetate phase was transferred into the liquid-liquidextractor and extracted with water until the pH of water became neutral.Ethyl acetate phase and aqueous phase were separated and ethyl acetatephase was collected and concentrated in an Agitated thin film evaporator(ATFE) to form concentrated ethyl acetate extract. Ethyl acetateconcentrate was fed into vacuum stripper and dried under vacuum at above500 mm of mercury to form acid hydrolysis product of water treated 90%methanol extract of fresh leaves of Costus pictus D. Don (yield 75 g).

50 mg of the acid hydrolysis product (ethyl acetate concentrate) wasaccurately weighed and transferred into a 50 ml standard flask anddissolved in methanol and made up to the mark using methanol.

The dissolved sample was analysed on a Shimadzu Prominence HPLC. Column:Thermo Betasil, C18, 250×4.6 mm, 5 μ. Solvent system (isocratic):Methanol-Acetonitrile-Water (40:15:45 v/v/v) containing 1% acetic acid,flow rate 1 ml/min, detection at 368 nm.

Three peaks were detected at 368 nm and these corresponded to quercetin,kaempferol and isorhamnetin as confirmed by co-injection with referencestandards.

Alkali Hydrolysis

150 g of 90% methanol extract of fresh leaves of Costus pictus D. Don,which has low oxalic acid and/or oxalates content (Sample 2) wasweighed. 6 L of 2 M NaOH was added and the mixture was transferred intoan agitator vessel. The mixture was heated at 40° C. for half an hourwith constant stirring. After half an hour the mixture was cooled and pHof the mixture was adjusted to 3 by adding concentrated HCl. The mixturewas transferred into a liquid-liquid extractor and ethyl acetate wasadded. Ethyl acetate phase and aqueous phase were separated and ethylacetate phase was collected and concentrated in an Agitated thin filmevaporator (ATFE) to form concentrated ethyl acetate extract. Theconcentrated ethyl acetate extract was fed into vacuum stripper anddried under vacuum at above 500 mm of mercury to form alkali hydrolysisproduct of water treated 90% methanol extract of fresh leaves of Costuspictus D. Don (yield 75 g).

50 mg of the alkali hydrolysis product was accurately weighed andtransferred into a 50 ml standard flask. Next the alkali hydrolysisproduct was dissolved in methanol and the flask was made up to the markusing methanol. The dissolved sample was analyzed by HPLC, Column:Thermo Betasil, C18, 250×4.6 mm, 5 μ. Solvent system: Solvent A-1%acetic acid in water, solvent B-1% acetic acid/water/acetonitrile(2:68:30). Gradient: 0 min. 7% solvent B increased to 90% solvent B.Flow rate 1 ml/min. Hydroxycinnamic acids were detected by HPLC at 324nm and identified by co-injection with reference standards. Accordingly,p-coumaric acid, caffeic acid, ferulic acid and sinapic acid wereidentified after alkali hydrolysis of the extract.

EXAMPLE 2

30 Kg of Sample 2 was dissolved in minimum amount of methanol and about30 Kg of silica gel was added in vessel. The mixture was swirled untilthe methanol evaporated and only a dry powder remained. The dried powderwas transferred in to the top of the column, which was already wetpacked with silica gel (50 Kg) and hexane. Column was initially elutedwith hexane followed by methanol. Methanol fractions were collected andhexane fractions were discarded. Methanol fraction was concentrated inan Agitated thin film evaporator (ATFE) to form concentrated extract.Concentrate was fed into vacuum stripper and dried under vacuum at above500 mm of mercury to get dry powder. The yield of the powder was 430 gfrom a starting material of 30 kg. This powder was referred to as powderof silica purified 90% methanol extract of Costus pictus D. Don. Theextract has low oxalic acid and/or oxalates content [Sample3]. Oxalicacid content in silica purified 90% methanolic extract of Costus pictusD. Don was 3%. Oxalic acid was analyzed as in Example 1.

HPLC Analysis

HPLC analysis of Sample 3 was conducted as described in Example 1. TheUV data at 254/324 nm shows several peaks which correspond tohydroxycinnamic acid derivatives and flavonoids. Alkali hydrolysis andacid hydrolysis confirmed the presence of hydroxycinnamic acids andflavonoids respectively.

Acid Hydrolysis

150 g of Sample 3 was weighed and Acid hydrolysis was conducted as shownin Example 1. The final product was acid hydrolysis product of purified90% methanol extract of fresh leaves of Costus pictus D. Don (yield 75g).

The acid hydrolysis product of purified 90% methanol extract of freshleaves of Costus pictus D. Don was analysed by HPLC as in Example 1.

Three peaks were detected at 368 nm and these corresponded to quercetin,kaempferol and isorhamnetin as confirmed by co-injection with referencestandards.

Alkali Hydrolysis

150 g of Sample 3 was weighed and alkali hydrolysis was conducted asshown in Example 1. The final product was alkali hydrolysis product ofpurified 90% methanol extract of fresh leaves of Costus pictus D. Don(yield 75 g).

The alkali hydrolysis product was analysed by HPLC as in Example 1.Hydroxycinnamic acids were detected at 324 nm and identified byco-injection with reference standards. Accordingly, p-coumaric acid,caffeic acid, ferulic acid and sinapic acid were identified after alkalihydrolysis of the extract.

EXAMPLE 3

3000 Kg of fresh leaves of Costus pictus D. Don were extracted with 90%methanol as in Example 1. The product obtained was dry powder of 90%methanol extract of fresh leaves of Costus pictus D. Don (Yield 75 Kg).

Oxalic acid was removed from the powder of 90% methanolic extract ofCostus pictus by a method described in Example 1. The yield of thepowder obtained was 34 kg from a starting material of 75 kg. This powderwas referred to as powder of 90% methanol extract of Costus pictus D.Don and the extract has low oxalic acid and/or oxalates content. Oxalicacid content was about 8%.

The above obtained powder was dissolved in water and transferred into aliquid-liquid extractor and extracted with ethyl acetate. Two phaseswere formed-aqueous and ethyl acetate. Ethyl acetate phase was collectedand aqueous phase was again treated with ethyl acetate two more times inthe liquid-liquid extractor and at each time ethyl acetate phase wascollected. All the ethyl acetate phase were combined and concentrated inan Agitated thin film evaporator (ATFE) to form concentrated ethylacetate extract. Ethyl acetate concentrate was fed into vacuum stripperand dried under vacuum at above 500 mm of mercury to form dry powder.The yield of the powder was 30 kg from a starting material of 34 kg.This powder was referred to as powder of ethyl acetate extract of 90%methanol extract of Costus pictus D. Don. The extract had low oxalicacid and/or oxalates content (Sample 4). Oxalic acid content in ethylacetate extract of 90% methanolic extract of Costus pictus D. Don was1.5%. Oxalic acid was analyzed as in Example 1.

HPLC Analysis

The HPLC analysis of Sample 4 was conducted as in Example 1. The UV dataat 254/324 nm shows several peaks which correspond to hydroxycinnamicacid derivatives and flavonoids. Alkali hydrolysis and acid hydrolysisconfirmed the presence of hydroxycinnamic acids and flavonoidsrespectively.

Acid Hydrolysis

The acid hydrolysis of sample 4 was conducted as shown in Example 1. Theproduct obtained was acid hydrolysis product of ethyl acetate extract of90% methanol extract of fresh leaves of Costus pictus D. Don (yield 75g).

The acid hydrolysis product was analysed by HPLC as in Example 1. Threepeaks were detected at 368 nm and these corresponded to quercetin,kaempferol and isorhamnetin as confirmed by co-injection with referencestandards.

Alkali Hydrolysis

The alkali hydrolysis was conducted as shown in Example 1 by using 150 gof sample 4. The product obtained was alkali hydrolysis product of ethylacetate extract of 90% methanol extract of fresh leaves of Costus pictusD. Don (yield 75 g) and it was analysed by HPLC as in Example 1.

Hydroxycinnamic acids were detected at 324 nm and identified byco-injection with reference standards. Accordingly, p-coumaric acid,caffeic acid, ferulic acid and sinapic acid were identified after alkalihydrolysis of the extract.

EXAMPLE 4

25 Kg of sample 4 was dissolved in minimum amount of methanol and about25 Kg of silica gel was added in a vessel. The mixture was swirled untilthe methanol evaporated and only a dry powder remained. The Dried powderwas transferred into the top of the column, which was already wet packedwith silica gel (50 Kg) and hexane. Column was initially eluted withhexane followed by methanol. Methanol fractions were collected andhexane fraction was discarded. Methanol fraction was concentrated in anAgitated thin film evaporator (ATFE) to form concentrated extract.Concentrate was fed into vacuum stripper and dried under vacuum at above500 mm of mercury to get dry powder. The yield of the powder was 245 gfrom a starting material of 25 kg. This powder was referred to as powderof purified ethyl acetate extract of 90% methanol extract of Costuspictus D. Don. The extract had low oxalic acid and/or oxalates content(Samples). Oxalic acid content in purified ethyl acetate extract of 90%methanolic extract of Costus pictus D. Don was 0.2%.

Oxalic acid was analyzed as in Example 1.

HPLC Analysis

HPLC analysis of sample 5 was conducted as in Example 1. The UV data at254/324 nm shows several peaks which correspond to hydroxycinnamic acidderivatives and flavonoids. Alkali hydrolysis and acid hydrolysisconfirm the presence of hydroxycinnamic acids and flavonoids,respectively.

Acid Hydrolysis

150 g of Sample 5 was subjected to acid hydrolysis as shown inExample 1. 75 g of acid hydrolysis product of purified ethyl acetateextract of 90% methanol extract of fresh leaves of Costus pictus D. Donwas obtained.

The acid hydrolysis product was analysed by HPLC as in Example 1. Threepeaks were detected at 368 nm and these corresponded to quercetin,kaempferol and isorhamnetin as confirmed by co-injection with referencestandards.

Alkali Hydrolysis

Alkali hydrolysis of sample 5 was conducted as in Example 1.75 g ofalkali hydrolysis product of purified ethyl acetate extract of 90%methanol extract of fresh leaves of Costus pictus D. Don was obtained.Alkali hydrolysis product was analysed by HPLC as in Example 1.Hydroxycinnamic acids were detected at 324 nm and identified byco-injection with reference standards. Accordingly, p-coumaric acid,caffeic acid, ferulic acid and sinapic acid were identified after alkalihydrolysis of the extract.

EXAMPLE 5

Hydroxycinnamic acids are non-flavonoid phenolics characterized by theC6-C3 structure (Table 1). These compounds are abundant in plants andare used in both structural and chemical plant defense strategies.Derivatives of cinnamic acid are present in numerous vegetables andfruits. In Costus pictus D. Don extract p-coumaric, caffeic, ferulic andsinapic acids are found in conjugation with sugar or otherhydroxycinnamic acids or quinic acid (R₃).

TABLE 1 Structure of different hydroxycinnamic acid derivatives presentin extract of Costus pictus D. Don. Hydroxycinnamic acids R₁ R₂ R₃

p-Coumaric Caffeic Ferulic Sinapic H OH OCH₃ OCH₃ H H H OCH₃ H H H H

-   -   R1 is hydrogen (H), hydroxyl (OH), methoxy(OCH3) group depends        on the hydroxycinnamic acid present in the extract of Costus        pictus D. Don.    -   R2 is hydrogen (H), methoxy (OCH3) depends on the        hydroxycinnamic acid present in the extract of Costus pictus D.        Don.    -   R3 is hydrogen (H) in all the four hydroxycinnamic acid present        in the extract of Costus pictus D. Don.

Flavonoids are plant pigments widely distributed in nature. They arepolyphenolic compounds comprising fifteen carbons with two aromaticrings connected by a three-carbon bridge, hence C6-C3-C6 (Table 2). Theyare present in high concentrations in the epidermis of leaves and fruitsand have important and varied roles as secondary metabolites, beinginvolved in processes like UV protection, pigmentation, stimulation ofnitrogen-fixing nodules and disease resistance. Flavonols are the mostwidespread of the flavonoids. Quercetin, kaempferol and isorhamnetin,the main flavonols in Costus pictus D. Don extract, are found asO-glycosides and as free flavonols. Conjugation occurs most frequentlyat the 3 position of the C-ring, but substitutions can also occur at the5, 7, 4′, 3′ and 5′ positions. Mono- di- and triglycosides containingglucose/galactose and rhamnose are found.

Flavonols R₁ R₂ R₃

Kaempferol Quercetin Isorhamnetin OH OH OH H OH OCH₃ H H H

-   -   R1 is hydroxyl group (OH) in thee flavonols present in the        extract of Costus pictus D. Don.    -   R2 is hydrogen (H), hydroxyl (OH), methoxy (OCH3) depends on the        flavonols present in the extract of Costus pictus D. Don.    -   R3 is hydrogen (H) in the structure of the flavonols present in        the extract of Costus pictus D. Don.

High performance liquid chromatography (HPLC) Analysis

The extract of Costus pictus was analyzed by HPLC for the presence ofhydroxyl cinnamic acid and flavonoids.

The extract was analyzed on a Shimadzu Prominence HPLC using aPhenomenex C-18 column (250×4.6 mm), 5 μ, RT (25° C.), Sampleconcentration −1 mg/ml dissolved in MeOH: Water (1:1).

A gradient elution using solvents 5% ACN(acetonitrile) in water (solventA) and 95% ACN (acetonitrile) in water (solvent B) having 5 mM ammoniumacetate (pH 4) was used. The composition of B was changed from 5% to 50in 35 min, then to 80% in 45 min and maintained at 80% till 55 min andthen maintained at 5% from 56 to 60 min. A UV detector at 254 nm and 324nm was used to collect the data.

The UV data at 254 nm (FIG. 1) shows several peaks corresponding tohydroxycinnamic acid. UV data at 324 nm (FIG. 2) shows several peakswhich correspond to flavonoids. Alkali hydrolysis and acid hydrolysisconfirm the presence of hydroxycinnamic acids and flavonoids,respectively.

Alkali Hydrolysis

Alkali hydrolysis was carried out with 2M sodium hydroxide at 40° C. forhalf-an-hour, acidified with HCl to pH 3 and extracted with ethylacetate, concentrated and dried under vacuum. Hydroxycinnamic acids weredetected by high performance liquid chromatography (HPLC) at 324 nm andidentified by co-injection with reference standards. Accordingly,p-coumaric acid, caffeic acid, ferulic acid and sinapic acid wereidentified after alkali hydrolysis of the extract (FIG. 3). Thisindirectly confirms the presence of derivatives of these acids in Costuspictus D. Don.

HPLC conditions:—Column: Thermo Betasil, C18, 250×4.6 mm, 5 μ. Solventsystem: solvent A-1% acetic acid in water, solvent B-1% aceticacid/water/acetonitrile (2:68:30). Gradient: 0 min. 7% B increased to90% B. Flow rate 1 ml/min. Detection at 320 nm.

HPTLC finger print of Costus pictus D. Don after alkali hydrolysis isshown in FIGS. 4a and 4 b. FIG. 4a provides HPTLC finger print of Costuspictus D. Don extract after alkali hydrolysis in visible light and FIG.4b provides HPTLC finger print of Costus pictus D. Don extract afteralkali hydrolysis in UV light.

HPTLC finger print (FIG. 4a and FIG. 4b ) of Costus pictus extract afteralkali hydrolysis shows the presence of p-coumaric acid, caffeic acid,ferulic acid and sinapic acid (Track 5) against reference standardsferulic acid (Track 1), Sinapic acid (Track 2), p-coumaric acid(Track 4)and caffeic acid (Track 5).

Acid Hydrolysis

Acid hydrolysis was carried out by refluxing with 1N HCl for 4 hrsfollowed by extraction with ethyl acetate. The ethyl acetate extract waswashed with water until the pH was neutral, concentrated, dried undervacuum and analysed by High performance liquid chromatography (HPLC).Three peaks were detected at 368 nm (FIG. 5) and these corresponded toquercetin, kaempferol and isorhamnetin as confirmed by co-injection withreference standards. Kaempferol was isolated by column chromatographyand its proton

NMR (nuclear magnetic resonance) data matched with that reported inliterature.

TABLE 3 ¹H and ¹³C NMR data of Kaempferol isolated from the acidhydrolysis product. ¹H ¹³C ¹³C 6.10 (1H, d, H-6) 145.96, C-2  93.82, C-86.32 (1H, d, H-8) 135.59, C-3 160.51, C-9 6.91 (2H, d, H-3′,5′) 175.51,C-4 101.77, C-10 8.03 (2H, d, H-2′,6′) 156.43, C-5 121.73, C-1′  99.06,C-6 129.17, C-2′,6′ 167.30, C-7 115.43, C-3′,5′ 159.23, C-4′ H denotesHydrogen, C denotes carbon.

HPLC conditions:—Column: Thermo Betasil, C18, 250×4.6 mm, 5 μ. Solventsystem (isochratic): Methanol-Acetonitrile-Water (40:15:45 v/v/v)containing 1% acetic acid, flow rate 1 ml/min, detection at 368 nm. (Y-LLi et al, Molecules, 13, 1931 -4 (2008); X. Liu et al, Food Chem.109,909-915 (2008).)

Sugar Analysis

After separation of flavonoids from the acid hydrolysis experiment, theaqueous layer after neutralization was tested for sugars present in theextract of Costus pictus using High performance thin layerchromatography (HPTLC) using reference sugars glucose, ribose, Dgalactose, fructose and rhamnose. Glucose, galactose and rhamnose wereidentified in the aqueous layer of acid hydrolysed Costus pictusextract, thereby indicating the presence of conjugates of these sugarsin the extract (HPTLC FIG. 6a &6 b).

HPTLC finger print of aqueous layer obtained from the acid hydrolysisexperiment of Costus pictus D. Don was shown in FIGS. 6a and 6b . FIG.6a provides HPTLC finger print of aqueous layer obtained from the acidhydrolysis experiment of Costus pictus D. Don extract in visible lightand FIG. 6b provides HPTLC finger print of aqueous layer obtained fromthe acid hydrolysis experiment of Costus pictus D. Don extract in UVlight.

HPTLC finger print (FIG. 6a and FIG. 6b ) of aqueous layer of Costuspictus extract after acid hydrolysis shows the presence of sugars likeglucose, galactose and rhamnose (Track 4) against reference standardsglucose (Track 1), ribose (Track 2), D-galactose (Track 3),

D-fructose (Track 4) and D-rhamnose (Track 7).

EXAMPLE 6

Liquid chromatography-mass spectrometry (LCMS) Analysis

The extract was subjected to LCMS analysis on a Waters Quattro II triplequadrupole mass spectrometer having Waters Alliance HPLC systemconnected to it. The column used was Thermo-ODS-2 (250×4.6 mm, 5 μ). Agradient elution using solvents 5% ACN (acetonitrile) in water (solventA) and 95% ACN (acetonitrile) in water having 5 mM ammonium acetate (pH4) was used. The composition of B was changed from 5% to 50 in 35 min,then to 80% in 45 min and maintained at 80% till 55 min and thenmaintained at 5% from 56 to 60 min. A photodiode array detector (PDA)detector (200-650 nm) was used to collect the high performance liquidchromatography (HPLC) data. The mass spectra were scanned in the rangeof 100-1000 Da (dalton) in 2s. The Electrospray Ionisation (ESI)capillary was set to 3.5 kV (kilo volt) and the cone voltage was 40 V.Dry nitrogen was used as the nebulizer (10 litre per hour) and dryinggas (250 litre per hour).

The source temperature was 80° C.

As photodiode array detector (PDA) detector was available the UV datacould be examined at different wavelengths. The chromatograms at 324 nmand 370 nm are given in FIGS. 7 & 8 respectively. The major constituentsappear to be hydroxycinnamic acid derivatives (at 324 nm) and flavonoidglycosides (at 370 nm).

Summary of the Tables and Figures

Hydroxycinnamic acid derivatives

The Liquid chromatography-mass spectrometry (LCMS) chromatogram at 324nm given in FIG. 9 clearly shows a number of components, mainlyhydroxycinnamic acid derivatives.

These could be tentatively identified from the MS data given in Table 4.It is clear that the coumaric acid is part of many of theseconstituents. There are at least seven components of the chlorogenicacid family. These are coumaroyl quinic acids (CQA), an example of whichis given below.

5-p-coumaroyl quinic acid (5-pCoQA)

TABLE 4 Identification of components of hydroxycinnamic acid from themass spectral data. S. No RT M. Wt MS Data (m/z) Remarks 1 5.48 342 360,342, 325, Caffeoyl hexose/Disaccharide 163, 145, 127 2 5.48 324 342,325, 307, Dehydrosugar/Disaccharide 289, 271, 163, 145 3 7.68 288 327,311, 306, Trihydroxyphenyl hexose 271, 253 4 9.58 356 374, 357, 339,Coumaric acid conjugate of 147 hexaric acid 5 10.60 356 374, 357, 227,Coumaric acid conjugate of 177, 147 hexaric acid 6 11.17 338 356, 339,207, Coumaroyl quinic acid 147 7 13.21 338 356, 339, 147 ″ 8 13.58 370388, 371, 225, Sinapoyl rhamnose/dimer of 207 sinapic acid & cinnamicacid 9 15.42 386 404, 387, 225, Sinapoyl rhamnose/dimer of 207 sinapicacid & coumaric acid 10 15.90 338 356, 339, 207, Coumaroyl quinic acid147 11 16.98 338 356, 339, 147 ″ 12 18.48 338 356, 339, 147 ″ 13 19.04338 356, 339, 147 ″ 14 19.89 338 356, 339, 147 ″ RT—Retention time M.Wt—Molecular weight MS data—Mass spectrum data

Flavonoid Derivatives

Flavonoids are characterized by strong absorption at around 260 and340-370 nm. Hence Photodiode array detector (PDA) data at 370 nm shouldbe specific for flavonoids. The data shown in FIG. 10 indicate thepresence of several flavonoid derivatives. At least 16 components couldbe detected and tentatively identified (Table 5). The fragment ionsresulting from elimination of the sugar units clearly indicate thesequence of the sugar units in these glycosides. For example, thecomponent having parent ion at m/z 757 correspond to quercetintriglycoside having a hexose and two rhamnose units. The sugar sequencecould be similarly identified for all the other flavonoid glycosidecomponents as shown in Table 5. All of them showed UV absorption maximaat around 260 and 350 nm confirming flavonoid glycosides. The chemicalformulae of some of the flavonoid glycosides were obtained from accuratemass measurements of the parent ions (Table 6).

Acid hydrolysis of the extract gave quercetin, kaempferol andisorhamnetin as the aglycones. Their identities were confirmed byco-injection with standard reference compounds on HPLC. The presence ofgalactose, glucose and rhamnose has also been confirmed by sugaranalysis following acid hydrolysis.

TABLE 5 Identification of components of flavonoids from mass spectraldata. 16 components are detected and identified as flavonoid glycosidesand free form. M. S. No RT Wt MS Data (m/z) λ_(max(wavelength)) Remarks1 15.73 756 757, 611, 465, 303 256, 352 Quer-Hex-Rha-Rha 2 17.13 740741, 595, 449, 287 265, 338 Kaem-Hex-Rha-Rha 3 17.25 770 771, 625, 449,317 266, 338 IsoR-Hex-Rha-Rha 4 17.34 610 611, 465, 303 260, 351Quer-Hex-Rha 5 17.61 770 771, 625, 479, 317 260, 346 IsoR-Hex-Rha-Rha 618.14 610 611, 465, 303 260, 346 Quer-Hex-Rha 7 18.95 594 595, 449, 287265, 347 Kaem-Hex-Rha 8 19.17 624 625, 479, 317 260, 347 IsoR-hex-Rha 919.32 464 465, 303 260, 352 Quer-Hex 10 20.07 594 595, 449, 287 265, 342Kaem-Hex-Rha 11 20.35 624 625, 479, 317 260, 352 IsoR-Hex-Rha 12 21.34448 449, 287 265, 346 Kaem-Hex 13 21.65 478 479, 317 265, 346 IsoR-Hex14 28.75 302 303 265, 363 Quercetin 15 33.37 286 287 265, 363 Kaempferol16 34.00 316 317 265, 363 Isorhamnetin RT—Retention time, M.Wt—molecular weight, MS data—mass spectra data Quer—Quercetine,IsoR—Isorhamnetin, Kaem—Kaempferol, Hex—Hexose, Rha—Rhamnose

TABLE 6 The chemical formulae of some of the flavonoid glycosidesobtained from accurate mass measurements of the parent ions. NominalExact mass Exact mass mass (calculated) (measured) Error (ppm) Formula 1755 755.2035 755.2028 −0.9 C₃₃H₃₉O₂₀ 2 739 739.2086 739.2080 −0.8C₃₃H₃₉O₁₉ 3 593 593.1506 593.1505 −0.2 C₂₇H₂₉O₁₅ 4 463 463.0877 463.0873−0.9 C₂₁H₁₉O₁₂ 5 623 623.1612 623.1612 0 C₂₈H₃₁O₁₆

The flavonoids appear to be mainly tri-, di- and monoglycosides ofquercetin, kaempferol and isorhamnetin along with small amounts of thefree flavonols. All tri- and diglycosides containglucose/galactose/rhamnose whereas the monoglycosides contain onlyglucose or galactose.

EXAMPLE 7

Evaluation of effect of different fractions of fresh leaves of Costuspictus D. Donon streptozotocin-induced Type 1 diabetic model in Wistarrats.

The study was conducted in adult male Wistar rats weighing 200-250 gmfor a period of 40 days (30 days of treatment and 10 days of induction).The animals were randomly divided into seven groups comprising six each.Diabetes was induced in all the groups except the normal control groupby the intra peritoneal administration of streptozotocin at 50 mg/kg.After the induction of diabetes, one group was kept as diabeticuntreated control and a positive control was maintained in which theanimals were treated with standard drug, glibenclamide at 3 mg/kgorally. All the other diabetic induced groups were treated withdifferent extracts at 300 mg/kg orally. The effect of the differentfractions was assessed by measuring the fasting blood glucose level,post prandial blood glucose level, serum insulin levels and glycosylatedHb. The anti hyperlipidemic effect was assessed by the estimation oftotal cholesterol, triglyceride, LDL cholesterol, HDL cholesterol, VLDL,muscle glycogen and liver glycogen. The antioxidant effects of thedifferent extracts were assessed by the measuring TBARS, superoxidedismutase (SOD), catalase, reduced glutathione (GSH) and glutathioneperoxidase levels (GPx). TBARS was determined by TBA assay (Ohkawa etal., 1979), SOD was measured using the nitroblue tetrazolium reductionmethod (Kakkar et al., 1984), Catalase was assayed using the methoddescribed by Aebi (1974). GPx was measured as described by Flohe andGunzler (1984), GSH was measured as per the method of Jollow et al.,(1974). Histopathological examination of pancreas was conducted toassess the antidiabetic effect of the different fractions of Costuspictus D. Don.

The experimental design was as follows:

Group 1—Normal control.

Group 2—Diabetic control—untreated diabetic animals.

Group 3—Positive control—diabetic animals treated with glibenclamide at3 mg/kg orally.

Group 4—diabetic animals treated with methanolic extract of fresh leavesof Costus pictus D. Don, the extract has low oxalic acid and/or oxalatescontent (Sample 2 prepared as per Example 1) at 40 mg/kg orally.

Group 5—diabetic animals treated silica purified ethyl acetate extractof 90% methanolic extract of fresh leaves of Costus pictus D. Don(Sample 5 prepared as per Example 4) and the extract has low oxalic acidand/or oxalates content at 40 mg/kg orally.

Group 6—diabetic animals treated with ethyl acetate extract of 90%methanol extract of fresh leaves of Costus pictus D. Don (Sample 4prepared as per Example 3) and the extract has low oxalic acid and/oroxalates content at 40 mg/kg orally.

Group 7—diabetic animals treated with methanolic extract of fresh leavesof Costus pictus D. Don. (Sample 1 prepared as per Example 1) at 40mg/kg orally.

Results:

Blood Glucose Level

The results of the study are presented in Table. 1. The differentfraction of Costus pictus D. Don showed a reduction in the blood glucoselevel on day 15 and day 30. The reduction in blood glucose level showedby group 5 having low oxalic acid and/or oxalates content was more thanthe other treated groups. 90% methanol extract of Costus pictus D. Donwith oxalic acid showed 3.45 times reduction in blood glucose level. 90%methanol extract of Costus pictus D. Don having low oxalic acid and/oroxalates content showed 4.55 times reduction in blood glucose level instreptozotocin-induced diabetes.

TABLE 1 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) and methanolic extract with oxalic acid (Group 7) on bloodglucose levels (mg/dl) in streptozotocin-induced diabetes. Groups 0^(th)day 15^(th) day 30^(th) day Group 1 70 71 71 Group 2 320 315 420 Group 3321 126 71 Group 4 325 185 71.5 Group 5 322 175 70.1 Group 6 323 17870.6 Group 7 320 183 92.5

Oral glucose tolerance test (OGTT) was performed following a glucosechallenge of 2.5 g/kg by oral gavage. Blood glucose was recorded at 30min (PPG₁), 60 min (PPG2), 120 min (PPG₃) and 240 min (PPG₄), after theglucose challenge on day 1, day 15 and day 30 of the study.

The results of the study are presented in Table 2. The differentfraction of Costus pictus D. Don showed a reduction in the PPG levelafter 240 min. The reduction in PPG showed by group 5 having low oxalicacid and/or oxalates content was more than the other treated groups.

TABLE 2 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) and methanolic extract with oxalic acid (group 7) on postprandial glucose levels. PPG2. Groups PPG1. 1 PPG1. 15 PPG1. 30 PPG2. 1PPG2. 15 30 Group 1 268.2 182.4 228.4 128.6 132.1 149.6 Group 2 511.6330.8 395.4 499.3 345.8 402.1 Group 3 522.8 318.3 232.1 478.4 217.4240.4 Group 4 318.4 219.9 227.8 184.8 134.2 230.8 Group 5 320.5 201.4217.4 240.8 102.4 262.4 Group 6 328.4 208.4 232.5 252.1 110.4 298.1Group 7 325.3 224.8 292.1 272.4 128.9 288.1 PPG4. Groups PPG3. 1 PPG3.15 PPG3. 30 PPG4. 1 PPG4. 15 30 Group 1 92.8 95.6 110.7 73.4 71.8 75.8Group 2 378.8 282.8 323.8 332.1 235.9 281.4 Group 3 332.1 115.3 177.3201.9 104.8 69.8 Group 4 141.3 98.6 90.2 102.1 70.8 73.6 Group 5 134.393.6 91.8 99.7 70.6 72.8 Group 6 148.2 96.4 92.4 101.4 71.2 73.4 Group 7201.4 104.8 106.4 110.5 90.8 95.2

Lipid Profile and Tissue Glycogen Levels

The results of the study on the effect of lipid profile and tissueglycogen levels are presented in Table.3 and Table.4 respectively. Theresults of total cholesterol, triglycerides, LDL and VLDL showed areduction when compared to the diabetic untreated control. High DensityLipoproteins values increased in the sample treated groups when comparedto diabetic untreated control. An increase in the muscle and liverglycogen levels were observed in the sample treated groups when comparedto diabetic untreated group. The effect of different fraction on lipidprofile and tissue glycogen levels was more in group 5.

TABLE 3 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) and ethanolic extract with oxalic acid (group 7) on totalcholesterol, triglyceride, LDL cholesterol, HDL cholesterol, VLDLlevels. TC TG LDL HDL VLDL Groups (mg/dl) (mg/dl) (mg/dl) (mg/dl)(mg/dl) Group 1 121 98 49 52 20 Group 2 241 196 181 20 40 Group 3 128100 56 52 20 Group 4 130 103 51 58 21 Group 5 128 95 49 60 19 Group 6129 100 50 59 20 Group 7 140 110 59 48 28

TABLE 4 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) and methanolic extract with oxalic acid (group 7) on muscleglycogen and liver glycogen levels. Muscle glycogen Liver glycogenGroups (mg/g tissue) (mg/g tissue) Group 1 10 48 Group 2 2 20 Group 3 846 Group 4 9.0 44 Group 5 9.3 47 Group 6 9.2 47 Group 7 8.3 41

Insulin and Glycosylated Hb Levels

The effect of the different extracts on insulin and glycosylated Hblevels is presented on Table 5. Sample treated groups showed an increasein the insulin level and a decrease in the glycosylated hameoglobinlevel when compared to diabetic untreated group. The effect was more ingroup 5.

TABLE 5 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) and methanolic extract with oxalic acid (group 7) on insulinand glycosylated Hb levels. Glycosylated Hb Groups Insulin level (μU/ml)(% total Hb) Group 1 16 0.5 Group 2 7 0.9 Group 3 13 0.5 Group 4 15 0.5Group 5 15.4 0.5 Group 6 15.2 0.5 Group 7 14.1 0.6

Antioxidant Enzymes

The effect of the extracts on antioxidant enzyme levels is presented inTable.6. The antioxidant enzyme levels of SOD, catalase, GSH and GPxwere increased in the sample treated groups and a reduction in the TBARSlevel was observed in all the sample treated groups when compared to thediabetic untreated group. The effect on the antioxidant enzymes andTBARS were more in group 5.

TABLE 6 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) and methanolic extract with oxalic acid (group 7) onantioxidant enzyme levels. Catalase (nM GPx SOD H2O2 (U/mg TBARS (mM(U/mg decomposed/ GSH of Groups MDA/100 g) protein) s/g (mg/100 g)protein) Group 1 0.66 5.04 66.45 49.68 11.23 Group 2 2.01 2.16 40.1222.45 3.96 Group 3 0.80 2.96 59.36 38.12 7.54 Group 4 0.91 4.21 63.2545.12 9.35 Group 5 0.95 4.60 64.12 48.32 9.78 Group 6 0.93 4.40 63.8947.88 9.60 Group 7 0.85 3.99 61.57 43.87 8.48

Pancreatic Histopathology

Histopathological examination of the pancreas of normal group revealednormal architecture. Administration of streptozotocin decreased thenumber of β cells and the untreated diabetic group revealed shrunkenislets with degenerative necrosis. In the sections of group 3, group 4,group 5, group 6 and group 7 islets appeared less shrunken than theuntreated diabetic control group and increased number of β cells wasalso observed. There were also a reduction in the number of initiatedlymphocytes and macrophages. The histopathological observations werepredominant in group 5. The mean number of islets in group 1 was 36. Ingroup 2, the mean number of islets was 5 while in group 3 it was 18.66.The mean number of islets in sample treated groups ie, Groups 4, 5, 6 &7 were 18, 22.83, 19.66 and 17.11 respectively. This shows that therewas regeneration of the damaged islet cells in the treated groups, themaximum of which was seen in group 5. Moreover, on immune histochemistrygroups 4 (FIG. 14), 5 (FIG. 15), 6 (FIGS. 16) and 7 (FIG. 17) showedintensely stained beta cells and an increase in the number of beta cellsin the islets compared to both diabetic control as well as the normalcontrol. The beta cells in groups 2 were extremely sparse. The betacells in group 3 were moderately stained and comparable to group 1.

EXAMPLE 8

Evaluation of effect of different fractions of Costus pictus D. Don onhigh fat diet-induced insulin resistance model with C57/BL 6J mice.

The study was designed to evaluate the effect of different fractions ofCostus pictus D. Don on high fat-induced resistance Type 2diabetic model(HFD). Six week-old C57B1/6J mice were allowed free access to food andwater. They were housed under identical standard conditions. The animalswere kept for acclimatization for a period of 7 days. After 7 days, themice were randomly divided into six groups of six mice each. The normalcontrol group was treated with normal regular diet and the rest of theanimals were fed with high fat diet for 6 weeks. After 6 weeks,methanolic extract of fresh leaves of Costus pictus D. Don, and theextract had low oxalic acid and/or oxalates content, silica purifiedethyl acetate extract of methanolic extract of fresh leaves of Costuspictus D. Don where the extract had low oxalic acid and/or oxalatescontent and ethyl acetate extract of 90% methanolic extract of freshleaves of Costus pictus D. Don where the extract had low oxalic acidand/or oxalates content were administered at 40 mg/kg p.o. for 6consecutive weeks. The experimental design was as follows:

Group 1—Normal control treated with normal regular diet.

Group 2—HFD treated group for 6 weeks.

Group 3—HFD for 6 weeks and Standard drug treated group for 6weeks—metformin.

Group 4—HFD for 6 weeks and methanolic extract of fresh leaves of Costuspictus D. Don where the extract had low oxalic acid and/or oxalatescontent (Sample 2 prepared as per example 1) at 40 mg/kg orally for 6weeks.

Group 5—HFD for 6 weeks and silica purified ethyl acetate extract of 90%methanolic extract of fresh leaves of Costus pictus D. Don where theextract had low oxalic acid and/or oxalates content (Sample 5 preparedas per example 4) at 40 mg/kg orally for 6 weeks.

Group 6—HFD for 6 weeks and ethyl acetate extract of 90% methanolextract of fresh leaves of Costus pictus D. Don where the extract hadlow oxalic acid and/or oxalates content (Sample 4 prepared as perexample 3) at 40 mg/kg orally for 6 weeks.

Observations

Body weights were determined biweekly. At the end of experimentalperiod, blood samples were collected to determine plasma glucose,insulin, leptin, glycosylated hemoglobin, triglycerides, totalcholesterol, LDL, HDL and VLDL.

Body Weight

The results of the body weight of the animals are displayed in Table. 1.At the end of 6 weeks, the mean body weight of all the animals increasedfrom 15 g to nearly 60 g. After the end of 12 weeks, the sample treatedgroups showed a reduction in the body weight. The effect was almostsimilar in all the treated groups except metformin treated group. Amongthe sample treated groups (group 4, 5 & 6), group 5 showed similareffect to the standard drug (metformin) treated group.

TABLE 1 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) on body weight. Body weight at biweekly interval (in grams)Groups 0 2 4 6 8 10 12 Gr. 1 15 18 22 25 28 32 35 Gr. 2 15 30 45 60 6058 56 Gr. 3 15 30 46 61 48 35 35 Gr. 4 13 28 44 62 52 40 39 Gr. 5 15 3046 62 48 38 35 Gr. 6 15 30 46 61 50 39 37

Blood Glucose

The results of the blood glucose level are depicted in Table.2. Theresults showed a reduction in the blood glucose level of all the sampletreated groups (group 4, 5 & 6). The reduction in blood glucose activitywas high in group 5.

TABLE 2 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) on blood glucose level. Groups Blood glucose (mg/dl) Gr. 1. 80Gr. 2. 229 Gr. 3. 100 Gr. 4. 145 Gr. 5. 140 Gr. 6. 141

Lipid Profile:

The results of the total cholesterol, triglycerides, LDL, HDL and VLDLare given in Table.3. The TC level of HFD treated group was high. Allthe sample treated groups showed a reduction in TC level. Similarly TG,LDL and VLDL showed higher values in HFD treated group. The LDL valuesof HFD treated group were low. But all the sample treated groups showeda reduction in the TG, LDL, VLDL and an increase in HDL. Among thesample treated groups (group 4, 5 & 6), group 5 showed a reduction inthe lipid profile which was almost similar to the standard drug(metformin) treated group.

TABLE 3 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) on lipid profile in high fat diet induced diabetes mellitus.TC TG LDL HDL VLDL Groups (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) Gr. 1.120 30 24 90 6 Gr. 2. 180 65 102 65 13 Gr. 3. 122 30 17 99 6 Gr. 4. 12420 26 94 4 Gr. 5. 120 30 17 97 6 Gr. 6. 122 25 21 96 5

Serum Insulin Levels

The results of serum insulin and glycosylated hemoglobin are shown inTable.4. The HFD treated group showed an increase in insulin values. Onthe other hand, the sample treated groups (group 4, 5 & 6) showed areduction in the serum insulin and glycosylated hemoglobin values. Amongthe samples treated group, the effect is more in group 5 which showed amore similar activity to the standard drug treated group.

TABLE 4 Effect of methanolic extract having low oxalic acid and/oroxalates content (group 4), ethyl acetate extract of 90% methanolextract having low oxalic acid and/or oxalates content (group 6) and itssilica purified part having low oxalic acid and/or oxalates content(group 5) on serum insulin, leptin and glycosylated hemoglobin levels inhigh fat diet induced diabetes mellitus. Insulin Glycosylated Hb Groups(ng/ml) (% of total Hb) Gr. 1. 0.7 0.5 Gr. 2. 3.8 1.0 Gr. 3. 1.7 0.5 Gr.4. 2.2 0.6 Gr. 5. 1.7 0.5 Gr. 6. 2.0 0.5

EXAMPLE 9

In vitro insulin secretogogue effect of the different fractions ofCostus pictus D. Don using isolated mouse islets.

Procedure

I. Animals

Four to six weeks old male mice (Swiss albino strain; weighing 25±2 g)used for the study were maintained in the optimum environmentalconditions.

II. Isolation of Islets

Pancreata isolated from animals which were euthanized with CO₂anesthesia according the method of Shewadae et al., (1999) was removedaspectically. Washed thrice with Hank's Balanced Salt Solution (HBSS)and were subjected to collagenase digestion at 37° C. The pancreata wasincubated in the dissociation medium (Dulbecco's modified minimumessential medium (DMEM) supplemented with collagenase, Soybean TrypsinInhibitor and Bovine Serum Albumin fraction V) for 10 minutes. Theprocedure was repeated thrice and the dissociation was stopped byaddition of chilled DMEM with 10% fetal bovine serum to the pancreaticdigest. The mixture was centrifuged at 200×g for 10 minutes and thesupernatant was discarded. After two washings with DMEM, the pellet wasseeded in culture flasks containing DMEM pH-7.2 supplemented with 10%fetal bovine serum. The flasks were incubated at 37° C. in a CO₂incubator.

III. Assessment of Islet Viability and Specificity

The viability of the islets was checked by Trypan blue dye exclusiontest (Warburton and James, 1995) and this estimate had shown >98% of thecells were viable.

IV. Preparation of the Extracts and Standards

The extracts and glibenclamide (10 nM) were dissolved in HBSS atrequired concentrations. The HBSS alone served as negative control andglibenclamide served as positive control. Three groups were givenextract of Costus pictus D. Don having low oxalic acid and/or oxalatescontent as follows

Group 1—90% methanolic extract of fresh leaves of Costus pictus D. Donwhere the extract had low oxalic acid and/or oxalates content (Sample 2prepared as per example 1).

Group 2—silica purified ethyl acetate extract of 90% methanolic extractof fresh leaves of Costus pictus D. Don where the extract had low oxalicacid and/or oxalates content. (Sample 5 prepared as per example 4)

Group-3 ethyl acetate extract of 90% methanolic extract of fresh leavesof Costus pictus D. Don where the extract had low oxalic acid and/oroxalates content. (Sample 4 prepared as per example 3)

V. Assessment of Insulin Secretogogue Effect In Vitro

The islets were hand-picked under sterile conditions and islets wereplaced in one well of 24 well plate in DMEM containing 10% FBS. After 24hours of recovery period, the spent medium was removed and the cellswere washed with HBSS. The cells were given an initial incubation in 1mL of HBSS for 30 minutes at 37° C. Then the islets were incubated withvarious concentrations of test material (25-200 μg/mL, at five doses)and positive control glibenclamide (10 nM). The islets were thenincubated for at 37° C. for 30 minutes and 50 μL of supernatants werecollected. All the samples were assayed for insulin content by ELISAmethod. The experiments were performed in triplicates.

Results

TABLE 5 In vitro insulin secretogogue effect of the extract E2 onisolated pancreatic islets Zero minutes Thirty minutes Concentration ofConcentration of Groups insulin (ng/mL) insulin (ng/mL) Cells alone 0.41.53 Glibenclamide (10 nM) 0.97 3.77 Extract (25 μg/mL) Group 1 0.332.50 Extract (25 μg/mL) - group 2 0.69 3.12 Extract (25 μg/mL) - group 30.41 3.01 Extract (50 μg/mL) - group 1 0.49 3.17 Extract (50 μg/mL) -group 2 0.59 4.11 Extract (50 μg/mL) - group 3 0.59 3.55

Inference

In the assessment of insulin secretogogue effect, at basal level therewas no significant variations in the insulin levels. At 25 μg/mL extractconcentration there was an increase in the level of insulin released inthe medium. At 50 μg/mL concentration, the release was highlysignificant and comparable with that of positive control(glibenclamide). The increase in the insulin concentration was higher ingroup 2.

Other modifications and variations to the invention will be apparent tothose skilled in the art from the foregoing disclosure and teachings.Thus, while only certain embodiments of the invention have beenspecifically described herein, it will be apparent that numerousmodifications may be made thereto without departing from the spirit andscope of the invention.

What is claimed is:
 1. A composition comprising an extract of Costuspictus D. Don, the extract comprising hydroxycinnamic acid derivativesand flavonols; and, wherein a total of oxalic acid and oxalates contentin the extract is less than 15%.
 2. The composition of claim 1, whereinthe flavonols comprise mono-flavonols, di-flavonols, flavonoltriglycosides and flavonols in free form.
 3. The composition of claim 1,wherein the hydroxycinnamic acid derivatives comprise caffeic acid,p-coumaric acid, ferulic acid and sinapic acid.
 4. The composition ofclaim 1, wherein the flavonols comprise quercetin, kaempferol,isorhamnetin, glucose derivatives of quercetin, glucose derivatives ofkaempferol, glucose derivatives of isorhamnetin, galactose derivativesof quercetin, galactose derivatives of kaempferol, galactose derivativesof isorhamnetin, rhamnose derivatives of quercetin, rhamnose derivativesof kaempferol, and rhamnose derivatives of isorhamnetin.
 5. A dosageform of the composition of claim 1, the dosage form selected from thegroup consisting of capsule, tablet, granule, sachet, powder, paste,ointment, infusion, injection, ampoule, solution, suspension, emulsion,pill, sustained release formulation and combinations thereof.
 6. Adosage form of the composition of claim 1 further comprising fillers,the fillers selected from the group consisting of lactose, spray driedlactose, starch, dibasic calcium phosphate, tribasic calcium phosphate,microcrystalline cellulose, hydroxy propyl methyl cellulose, and calciumcarbonate.
 7. A dosage form comprising a dosage in the range of about500 mg/day to 2000 mg/day of composition for administration to a humansubject.
 8. The composition of claim 1, wherein the extract of Costuspictus D. Don is selected from the group consisting of a) a methanolextract of Costus pictus D. Don comprising about 10% of oxalic acid; b)a methanol extract of Costus pictus D. Don comprising about 5% of oxalicacid; c) an ethyl acetate extract of Costus pictus D. Don comprisingabout 2.5% of oxalic acid; and, d) an ethyl acetate extract of Costuspictus D. Don comprising about 1% of oxalic acid.
 9. A compositioncomprising an extract of Costus pictus D. Don, the extract comprisingabout 10% of oxalic acid, the extract prepared by a method comprising:a) cleaning a starting material of Costus pictus D. Don; b) cutting theproduct of step (a); c) extracting the product of step (b) with 90%methanol; d) refluxing the product of step (c) for at the boilingtemperature (60-70° C.) of methanol to obtain a residue and asupernatant; e) filtering to separate the residue and the supernatant ofstep (d); concentrating the supernatant of step (e) to obtain aconcentrated methanol extract; g) drying the concentrated methanolextract of step (f) to obtain a powder of the methanol extract; h)mixing the powder of step (g) with water; i) heating the product of step(h) to obtain a residue and a supernatant; j) separating the residuefrom the supernatant; k) drying the residue to obtain a dry powdercomprising about 10% of oxalic acid.
 10. A composition comprising anextract of Costus pictus D. Don, the extract comprising about 5% ofoxalic acid, the extract prepared by a method comprising: a1) mixing thedry powder of step (k) of claim 9 with methanol and silica gel to obtaina mixture; a2) evaporating methanol from the mixture of step a1) toobtain a dry powder; a3) loading the dry powder of step a2) onto a wetpacked silica gel column to obtain an extract loaded silica gel column;a4) eluting the extract loaded silica gel column of step a3) with hexaneto obtain a silica gel column after hexane elution; a5) eluting thesilica gel column eluted with hexane of step a4) with methanol andobtaining a methanol eluate fraction; a6) concentrating the methanoleluate fraction to form a concentrated extract; a7) drying theconcentrated extract of step a6) to obtain a dry powder comprising about5% oxalic acid, and wherein the wet packed silica gel comprises silicagel and hexane.
 11. A composition comprising an extract of Costus pictusD. Don, the extract comprising about 2.5% of oxalic acid, the extractprepared by a method comprising: b1) dissolving the dry powder of stepk) of claim 9 in water to obtain a mixture; b2) extracting the mixtureof step b1) with ethyl acetate to obtain an aqueous phase and an ethylacetate phase; b3) concentrating the ethyl acetate phase of step b2) toobtain a concentrated ethyl acetate extract; b4) drying the ethylacetate extract under vacuum at above 500 mm of mercury to form a drypowder of the ethyl acetate extract comprising about 2.5% of oxalicacid.
 12. A composition comprising an extract of Costus pictus D. Don,the extract comprising about 2.5% of oxalic acid, the extract preparedby a method comprising: c1) mixing the dry powder of step b4) of claim11 with methanol and silica gel to obtain a mixture; c2) drying themixture of step c1) to obtain a dry powder: c3) loading the dry powderof step c2) onto a wet packed silica gel column to obtain an extractloaded silica gel column, wherein the wet packed silica gel columncomprises silica gel column in hexane; c4) eluting the extract loadedsilica gel column of step c3) with hexane to obtain a silica gel columnafter hexane elution; c5) eluting the silica gel column eluted withhexane of step c4) with methanol and obtaining a methanol eluatefraction; c6) concentrating the methanol eluate fraction of step c5) toform a concentrated extract; c7) drying the concentrated extract of stepc6) to obtain a dry powder comprising about 1% oxalic acid.